ABSTRACT The overall goal of this proposal is to understand calcium regulation in both cardiac and skeletal muscle at the atomic level. Calcium (Ca2+) is an essential messenger for muscle contractility, and its homeostatic balance is controlled by proteins embedded or peripheral to the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca2+-ATPase (SERCA) regulates diastole by translocating ~70% of the Ca2+ ions. In its unphosphorylated state, phospholamban (PLN) reduces SERCA's affinity for Ca2+, whereas phosphorylation of PLN at Ser16 re-establishes basal Ca2+ transport. Sarcolipin (SLN), a PLN homolog, regulates SERCA in both skeletal and cardiac muscle, keeping SERCA's activity within a physiological window. Like PLN, SLN is also modulated by ?-adrenergic stimulation through phosphorylation at Thr5, though the role of this phosphory- lation event in SERCA regulation is incompletely understood. Dysregulation of SERCA's function outside the physiological window by PLN and SLN leads to cardiomyopathies and heart failure. SLN is also proposed to play a crucial role in metabolism and skeletal muscle thermogenesis through uncoupling ATP hydrolysis and Ca2+ transport in SERCA. Though there is compelling biological evidence for this phenomenon, the molecular mechanisms by which SLN elicit the functional uncoupling of SERCA is unknown. In this competitive renewal, we will build on our previous discoveries and take new and exciting directions to understand the atomic details of PLN and SLN's allosteric regulation of SERCA and unveil the different roles of these endogenous inhibi- tors both in cardiac and skeletal muscle. In AIM1, We will focus on the structural analysis of SERCA regulation by two mutants, PLNP21G and PLNM20GP21G, which we designed and currently testing for gene therapy. We will study how these mutants mimic the phosphorylated state of PLN and tune SERCA activity. In AIM2, we will determine the role of SLN in non-shivering muscle thermogenesis. Finally, in AIM3, we will study the functional and structural role of a newly discovered regulator of the SERCA/PLN complex: HAX-1, which has an anti- apoptotic role in cardiomyocytes. These studies will be carried out using a combination of molecular biology, biochemical assays, thermocalorimetry, and spectroscopic methods (solution, solid-state NMR and fluores- cence) in synthetic and native membranes. To accomplish these specific AIMs, we have assembled a strong collaborative team, including Dave Thomas, Muthu Periasamy, Roger Hajjar, Evangelia Kranias, and Seth Robia. These scientists are at the forefront of the study of Ca2+ physiology and pathophysiology and have committed to a collaborative effort and timely data sharing plan that will propel our research and ensure sound biological and biomedical significance for our studies. The outcomes of this research will have a strong im- pact for understanding excitation-contraction coupling, muscle contractility, and thermogenesis, with significant implications for the design of alternative therapeutic approaches to counteract muscle disease.